Does modulation of organic cation transporters improve pralidoxime activity in an animal model of organophosphate poisoning?

OBJECTIVES: Pralidoxime is an organic cation used as an antidote in addition to atropine to treat organophosphate poisoning. Pralidoxime is rapidly eliminated by the renal route and thus has limited action. The objectives of this work were as follows. 1) Study the role of organic cation transporters in the renal secretion of pralidoxime using organic cation transporter substrates (tetraethylammonium) and knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻). 2) Assess whether sustained high plasma concentrations increase pralidoxime antidotal activity toward paraoxon-induced respiratory toxicity. SETTING: INSERM U705, Faculté de Pharmacie, Université Paris Descartes, 4 Avenue de l'Observatoire, 75006 Paris, France. SUBJECTS: Rodents: Knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻) and Sprague-Dawley rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In rats, the renal clearance of pralidoxime was 3.6-fold higher than the creatinine clearance. Pretreatment with tetraethylammonium (75 mg/kg) in rats or deficiencies in organic cation transporters 1 and 2 in mice (Oct1/2⁻/⁻) resulted in a significant increase in plasma pralidoxime concentrations. Lack of Oct3 did not alter plasma pralidoxime concentrations. The antidotal activity of pralidoxime (50 mg/kg intramuscularly) was longer and with greater effect, resulting in a return to normal values when administered to rats pretreated with tetraethylammonium. CONCLUSIONS: Pralidoxime is secreted in rats and mice by renal Oct1 and/or Oct2 but not by Oct3. Modulation of organic cation transporter activity increased the plasma pralidoxime concentrations and the antidotal effect of pralidoxime with sustained return within the normal range of respiratory variables in paraoxon-poisoned rats. These results suggest a promising approach in an animal model toward the increase in efficiency of pralidoxime. However, further studies are needed before these results are extended to human poisoning.

Reevaluation of indirect field stimulation technique to demonstrate oxime effectiveness in OP-poisoning in muscles in vitro.

Organophosphorus (OP) pesticides or nerve agents cause severe intoxication by inhibition of acetylcholinesterase, finally resulting in death due to respiratory failure. The phrenic nerve diaphragm preparation is considered as the classic model to investigate the effect of OP intoxications and oxime treatment at the neuromuscular junction. However, this preparation is unsuitable for larger species or for muscle strips from biopsies where no nerve is available for stimulation. An alternative technique is the indirect field stimulation of muscles containing intramuscular nerve branches only. The proposed method by Wolthuis et al. [Wolthuis, O.L., Vanwersch, R.A.P., Van Der Wiel, H.J., 1981. The efficacy of some bis-pyridinium oximes as antidotes to soman in isolated muscles of several species including man. Eur. J. Pharmacol. 70, 355-369] was modified and experimentally reevaluated in isolated mouse diaphragms. To confirm that electrical field stimulation technique induced muscle contraction only via the neuromuscular endplate the nicotinic antagonists pancuronium or d-tubocurarine (1microM) were given. In the presence of a nicotinic antagonist hardly any contraction was blocked after indirect field stimulation technique with very short pulses (5micros, <0.6A), in contrast to direct muscle stimulation (broader pulse width, or higher amplitude >0.6A). During paraoxon circumfusion (20min, 1micromol/l) muscle force generation by indirect stimulation was almost completely blocked. Restoration of paralyzed muscle function to 80% of initial values could be achieved after paraoxon wash out (20min) and circumfusion with obidoxime (1micromol/l, 20min). This data correspond quite well to data shown earlier when using conventional nerve stimulation techniques.

Paraoxonase and susceptibility to organophosphorus poisoning in farmers dipping sheep.

OBJECTIVES: Human serum paraoxonase (PON1) hydrolyses organophosphate pesticides (OPs) entering the blood circulation and tissue fluid thus limiting toxicity. The PON1 coding region has two polymorphisms involving the amino acids at position 55 (Lt<--M) and 192 (Qt<--R), giving rise to isoenzymes which differ in their catalytic rate for the hydrolysis of OPs. We therefore hypothesized that individuals inheriting low activity isoforms of PON1 would be more liable to report symptoms of OP toxicity. METHODS: We have therefore investigated the relationship between PON1 genetic polymorphisms and PON1 activity in farmers reporting chronic ill health which they attributed to OP exposure whilst sheep dipping (cases) and farmers who carried out similar activities, but remained well (controls). Diazoxon, paraoxon and phenylacetate were used as substrates for PON1. Diazoxon is the active metabolite of diazinon, the sheep dip most commonly used in the UK. RESULTS: Cases were found to be more likely to have the R192 allele ( 0.01) and to have the L55 allele ( 0.05) than the controls. This combination of R and L genotypes was associated with lower PON1 activity towards diazoxon in both cases and controls. Farmers in the lowest quintile for the rate of serum diazoxon hydrolysis had a greater risk of being a case i.e. of reporting ill health (odds ratio 2.47 (95% CI 1.35-2.82)), than the other four quintiles of diazoxon hydrolysis. The rate of serum hydrolysis of paraoxon was greatest in cases and controls with the R/L haplotype (both 0.001). CONCLUSIONS: The farmers reporting chronic ill health due to organophosphate exposure have a higher proportion of the PON1-192R polymorphism associated with lower rates of diazoxon hydrolysis and lower rates of diazoxon hydrolysis than the controls and that their ill health may be explained by a lower ability to detoxify diazoxon.

Comparing therapeutic and prophylactic protection against the lethal effect of paraoxon.

Prophylactic and therapeutic efficacy against organophosphorus (OP) intoxication by pralidoxime (2-PAM) and atropine were studied and compared with sterically stabilized long-circulating liposomes encapsulating recombinant organophosphorus hydrolase (OPH), either alone or in various specific combinations, in paraoxon poisoning. Prophylactic and therapeutic properties of atropine and 2-PAM are diminished when they are used alone. However, their prophylactic effects are enhanced when they are used in combination. Present studies indicate that sterically stabilized liposomes (SL) encapsulating recombinant OPH (SL-OPH) alone can provide much better therapeutic and prophylactic protection than the classic 2-PAM + atropine combination. This protection was even more dramatic when SL-OPH was employed in combination with 2-PAM and/or atropine: the magnitude of prophylactic antidotal protection was an astounding 1022 LD(50) [920 mg/kg (LD(50) of paraoxon with antagonists)/ 0.95 mg/kg (LD(50) of control paraoxon)], and the therapeutic antidotal protection was 156 LD(50) [140 mg/kg (LD(50) of paraoxon with antagonists)/0.9 mg/kg (LD(50) of control paraoxon)]. The current study firmly establishes the value of using liposome encapsulating OPH.

Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice.

Parathion hydrolase purified from Pseudomonas sp. was injected i.v. into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning. Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs. The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase. OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.

Electromyography in relation to end-plate acetylcholinesterase in rats poisoned by different organophosphates.

Organophosphate (OP) poisoning produces various forms of acute, subacute, or delayed neurotoxicity. We investigated in vivo the relationship between clinical, histochemical and electromyographic (EMG) parameters in rats at various stages of poisoning by paraoxon or fenthion. Paraoxon is acutely toxic, whereas fenthion produces more sustained AChE inhibition. Fenthion has been involved in a subacute type of OP-related neurotoxicity in patients, the so-called intermediate syndrome. The animals underwent serial EMGs, with single and repetitive nerve stimulation, and concomitant contralateral muscle biopsies to determine the end-plate acetylcholinesterase (AChE) activity. Repetitive activity (RA) after single nerve stimulation and decrements on repetitive nerve stimulation (RNS) were the major EMG findings in either type of poisoning, occurring in the initial and later stages of the poisoning, respectively. RA was highly correlated to fasciculations in acute, but not in prolonged intoxication. Amplitude decrements provoked by RNS occurred only in weak rats with severe end-plate AChE inhibition. The smallest amplitude occurred either at the second response with gradual improvement in the subsequent responses (decrement-increment phenomenon), or the amplitude decrease progressed up to the last response (decrement phenomenon). The decrement-increment phenomenon preceded the decrement phenomenon and occurred at a slightly less severe degree of AChE inhibition. Various types of impairment of neuromuscular transmission coexist, probably to a different extent at distinct stages of anticholinesterase poisoning.

Acute and subacute organophosphate poisoning in the rat.

The intermediate syndrome in organophosphate poisoning is clinically characterized by weakness in the territory of cranial nerves, weakness of respiratory, neck and proximal limb muscles, and depressed deep tendon reflexes. It occurs between the acute cholinergic crisis and the usual onset of organophosphate-induced delayed neurotoxicity. The weakness has been ascribed to muscle fiber necrosis. Fenthion has been the most common cause. This study assesses the occurrence of the necrotizing myopathy in rats in relation to the clinical course and the acetylcholinesterase (AChE) inhibition after poisoning with organophosphates representative for each of the major types of organophosphate-related neurotoxicity. Marked differences are noted in the duration of cholinergic symptoms and of AChE inhibition after either paraoxon and mipafox, or fenthion poisoning. The necrotizing myopathy begins shortly after the initial decline in AChE activity with all organophosphates studied. Maximal muscle involvement occurs within the first 2 days of the poisoning with all organophosphates studied. The myopathy is not aggravated by a further decline in AChE activity in fenthion poisoning. Our data argues against the monophasic necrotizing myopathy being the cause of the intermediate syndrome, and is suggestive of persistent AChE inhibition being involved.

Quinidine prevents paraoxon-induced necrotizing myopathy in rats.

Acute organophosphorus anticholinesterase poisoning induces a necrotizing end-plate myopathy in rats and patients. Acetylcholine (ACh) excess leads to prolonged synaptic currents and increased influx of cations including calcium through the postsynaptic ACh receptor channels with prolonged muscle membrane depolarization, excess calcium influx into the sarcoplasm, and ultimately muscle fiber necrosis. Quinoline derivatives such as quinidine induce or worsen pre- and postsynaptic disorders of neuromuscular transmission in humans, and are beneficial in patients suffering from a rare congenital myasthenic syndrome called the slow channel congenital myasthenic syndrome. These drugs correct the prolonged opening times of the mutated acetylcholine receptor channels in this myasthenic syndrome. We treated paraoxon-poisoned rats with 4 x 10 or 4 x 50 mg/kg of quinidine and assessed the severity of the necrotizing myopathy in gastrocnemius and diaphragm muscle biopsies. Fasciculations were decreased and the necrotizing myopathy was prevented in most treated rats, with absence of necrotic muscle fibers in most animals in the high-dose group. Survival was not different from untreated poisoned animals. A number of physiological mechanisms, including blocking of presynaptic voltage-gated sodium or calcium channels or inhibition of the postsynaptic ACh receptors channels may have contributed to the attenuation of the myonecrosis. The optimal dose and the drug of choice amongst the clinically available quinoline derivatives remains to be determined.

Oral treatment of organophosphate poisoning in mice.

OBJECTIVE: Organophosphates are used as pesticides, herbicides, and chemical warfare agents. Treatment of organophosphate poisoning is with intravenous atropine and pralidoxime in addition to supportive care. This study determined the efficacy of oral agents in preventing death from organophosphate poisoning. METHODS: The organophosphate paraoxon (8 mg/kg) was used in a murine model with lethality at four and 24 hours as an end point. For oral treatment, 15 male Balbc mice were given either atropine sulfate (4 mg/kg), or a combination of atropine sulfate (4 mg/kg) with pralidoxime (100 mg/kg), by oral gavage. A control group of 22 mice received water by oral gavage. Chi-square analysis was used to compare results in the different groups. RESULTS: Of the control group, six of 22 survived to four hours after paraoxon exposure. Of the exposed animals treated with oral atropine, eight of 15 survived to four hours. Of the exposed animals treated with a combination of atropine and pralidoxime, 13 of 15 survived to four hours. All animals surviving to four hours survived to 24 hours. The increased survival of animals in the atropine group relative to the control group was not significant (p = 0.09). Survival was significant in the group treated with atropine and pralidoxime relative to atropine alone (p = 0.02) and to the control group (p = 0.0002). All treated mice surviving at four hours were alive at 24 hours. CONCLUSIONS: Both oral atropine and a combination of oral atropine and pralidoxime improved survival, and combination therapy achieved statistical significance. Generalization of this result to other organophosphate pesticides, other doses of paraoxon, and other species cannot be made without further investigations.

Short-term effects of paraoxon and atropine on schedule-controlled behavior in rats.

The effects of lethal (2.0 mg/kg) and high sublethal (1.3 mg/kg) dosages of the organophosphate acetylcholinesterase (AChE) inhibitor paraoxon on FR10 performance rate was determined 1 and 2 days after intoxication. The lethal doses were antidoted with either centrally acting atropine sulfate (AS), or atropine methyl bromide (AMB) or atropine methyl nitrate (AMN), both quaternary salts and not expected to act centrally. AChE inhibition in the brain was about 35-60% on the second day after treatment. AS yielded a small transient depression in performance, while AMB and AMN yielded severe deficits, with incomplete recovery. Performance was depressed by 1.3 mg/kg paraoxon by 52% and 34% on days 1 and 2, respectively, while performance was more greatly depressed by the lethal dose, especially with the noncentrally acting antidotes: AS, 67 and 48%; AMB, 81 and 55%; AMN, 91 and 78%. However, a low dose of AS with 2 mg/kg paraoxon resulted in very severe, nonrecovering deficits. A lethal dose of the nonpersistent anti-AChE eserine sulfate, antidoted with a low dose of AS, yielded no deficits. Thus, a high level, acute intoxication with paraoxon yields behavioral deficits which are attenuated by high levels of a centrally acting muscarinic receptor antagonist. The paraoxon-induced performance deficits or their recovery do not correlate directly with AChE inhibition.

Sugar conjugates of pyridinium aldoximes as antidotes against organophosphate poisoning.

A series of pyridinium aldoximes having a sugar conjugated to the pyridine ring has been prepared as potential antidotes against organophosphate poisoning. The sugar residue was attached either directly through C-1 or C-6 of the pyranose ring or through a C3 bridge between the glycosyl group and the nitrogen atom of the pyridine moiety. Attachment of a sugar group to the oxime derivative seems to increase the bioavailability of the antidote. The clearance rate of the sugar conjugates was significantly lower than that of their non-sugar analogs and thus they were retained longer in the blood circulation. The sugar derivatives were more potent in decreasing paraoxon-induced hypothermia (which is regulated within the central nervous system) than N-methyl-2-pyridiniumaldoxime methanesulfonate, one of the most commonly used mono-oximes. The sugar analogs were also less toxic than the non-sugar analogs; some also displayed higher efficacy. The mechanism underlying the improved features of the sugar oximes, and the structural requirements in relation to the sugar attachment to the oxime function, are discussed.

[Participation of a distant cholinergic mechanism in the response of the vascular bed to intoxication by organophosphorus inhibitors of cholinesterase]. / Uchastie distantnogo kholinergicheskogo mekhanizma v reaktsii sosudistogo rusla na intoksikatsiiu fosfororganicheskimi ingibitorami kholinésterazy.

Poisoning with phosphacol causes dose--dependent decrease of blood flow speed in small vessels of mesoappendix. Administration of LD50 of phosphacole results in blood stagnation, simultaneous blood pressure fall, which leads to death of part of the animals. Electron microscopic study revealed the presence of acetyl and buthyryl cholinesterase in endotheliocytes of mesoappendicular capillaries, the activity of which was completely suppressed by administration of LD50 of phosphacol. 0,0-dimethyl-0 (2,2-dichlorvinyl) phosphate LD10 caused the damage of endotheliocyte surface. It was suggested that endothelial cholino-receptors that are activated through the rise of redundant acetyl-choline level in blood on the background of cholinesterase inhibition participate in the mechanism of pathological reactions described. Such variant of toxic effect was characterized as distant.

[The selection of the indices of antidote efficacy]. / Vybor pokazatelei éffektivnosti protivoiadiia.

The application of the following new indices of the efficacy of antidotes has been substantiated and discussed: the "antidotal potency" as a ratio of LD50 of a poison when an antidote used to LD99 in control intact animals, and the "index of solid protection" as a ratio of LD10 in the experiments to LD90 in controls. The two indices are true for changing the lethality curve slope, which makes it impossible to use the currently accepted "protection index" as a ratio of LD50 in experiments to LD50 in controls.

[Histamine blockers increase efficacy of specific antidote prophylaxis of intoxication by cholinesterase inhibitors in mice]. / Gistaminoblokatory povyshaiut éffektivnost' spetsificheskoi antidotnoi profilaktiki otravlenii myshei inhibitorami kholinésterazy.

Dimedrol and pipolphen, but not loratadine, increase efficacy of the atropine and dipiroxime treatment of mice--an antidote prophylaxis against their intoxication with phosphacol and aminostigmine. The results of experiments confirm a hypothesis that histamine participates in the formation of secondary toxicity reactions in the case of heavy poisoning with anticholinesterase agents.

[The antagonism of phosphorus-containing heterocyclics with the anticholinesterase agent Phosphacol (paraoxon)]. / Antagonizm fosforsoderzhashchikh geterotsiklov s antikholinésteraznym sredstvom fosfakolom (paraoksonom).

Effects of 15 derivatives of 2-R-oxo-(tioxo-3,5,5-trimethyl-1,2-oxaphospholan-3-ol (OPh) on lethality and protective action on cholinesterase (ChE) of mice brain and blood serum as a result of phosphacolum (paraoxonum) poisoning were investigated. Oxo and tioxo derivatives of OPh with O-methyl and O-ethyl, but not O-propyl and O-butyl radicals at phosphoric atom prevented the lethality of mice poisoned with phosphacolum and preserved brain and blood ChE of inhibition. Ethylic and phenylic OPh derivatives also prevented the lethality of poisoned animals, although without the protection of brain and blood plasma ChE. Combined application of OPh O-ethylic derivative with atropinum and dipiroxinum significantly potentiated antidotal effect m mice poisoned by phosphacolum.

[The early response of the blood lymphocytes in mice to phosphacol and its practical significance]. / Rannii otvet limfotsitov krovi myshei na fosfakol i ego prakticheskaia znachimost'.

Phosphacol drops applied to the eye of a mouse caused an immediate response of the blood lymphocytes. The number of phosphacol-immune rosette-forming and blast forms of lymphocytes increased. The response is specific since the formation of rosettes is blocked by the excess of phosphacol in vitro and does not occur when rausedyl-sensibilized erythrocytes are used. The formation of blast forms is intensified after small doses of phosphacol (10-9), but not rausedyl, are put into a suspension of blood cells. The technique proposed may be used for determination of phosphorus-containing compounds in the organism and the diagnosis of poisoning with them.

A conjugate of pyridine-4-aldoxime and atropine as a potential antidote against organophosphorus compounds poisoning.

A conjugate of pyridine-4-aldoxime and atropine (ATR-4-OX) was synthesized and its antidotal efficiency was tested in vitro on tabun- or paraoxon-inhibited acetylcholinesterase (AChE) of human erythrocytes as well as in vivo using soman-, tabun- or paraoxon-poisoned mice. Its genotoxic profile was assessed on human lymphocytes in vitro and was found acceptable for further research. ATR-4-OX showed very weak antidotal activity, inadequate for soman or tabun poisoning. Conversely, it was effective against paraoxon poisoning both in vitro and in vivo. All animals treated with 5 % or 25 % LD(50) doses of the new oxime survived after administration of 10.0 or 16.0 LD(50) doses of paraoxon, respectively. Based on the persistence of toxicity symptoms in mice, the atropine moiety had questionable effects in attenuating such symptoms. It appears that ATR-4-OX has a therapeutic effect related to the reactivation of phosphylated AChE, but not to receptor antagonization.

Muscle force and acetylcholinesterase activity in mouse hemidiaphragms exposed to paraoxon and treated by oximes in vitro.

The therapy of organophosphorus compound (OP) poisoning is still a challenge to clinical toxicologists. To alleviate peripheral respiratory failure oximes, e.g. obidoxime and pralidoxime, are used to reactivate inhibited acetylcholinesterase (AChE) with the intention to restore the disturbed neuromuscular function. In severe human OP poisoning the persistence of poison may counteract effective reactivation by oximes. Therefore, the study was designed to investigate the effect of the clinically used oximes obidoxime, pralidoxime and the experimental compounds HI 6 and HLö 7 in the presence of different paraoxon concentrations. The mouse phrenic nerve-diaphragm preparation was used as a functional model. After washout of paraoxon remarkably low concentrations of obidoxime or HLö 7 were sufficient for restoration of paraoxon-impaired muscle force. In the presence of paraoxon, obidoxime was the most effective oxime and therapeutically used concentrations (10-20microM) were able to restore muscle function even in the presence of 1microM paraoxon. HLö 7 was less effective, but superior to HI 6 and pralidoxime. Generally, a reactivation of AChE to about 30-40% of normal was sufficient for restoration of muscle force. Thus, the data presented strongly support the administration of appropriately dosed oximes, preferably obidoxime, in paraoxon-poisoned patients to restore paraoxon-impaired muscle force.

Pharmacokinetics and toxicodynamics of pralidoxime effects on paraoxon-induced respiratory toxicity.

Empirical studies suggest that the antidotal effect of pralidoxime depends on plasma concentrations with therapeutic effects associated with concentrations above 4 mg/l. The purpose of this study was to determine the pharmacokinetic-toxicodynamic (PK-TD) relationships for the antidotal effect of pralidoxime on paraoxon-induced toxicity in rats. Diethylparaoxon inactivation of whole-blood cholinesterase activity was studied both in vitro and in male Sprague-Dawley rats. Toxin-induced respiratory effects were measured via whole-body plethysmography in control and pralidoxime-treated animals (50 mg/kg im injection). In the in vitro analysis, cholinesterase reactivation by pralidoxime in blood-poisoned diethylparaoxon (10nM) was proportional to the logarithm of drug concentrations. A mechanism-based TD model was developed, which well described the inhibition of cholinesterases by diethylparaoxon and reactivation with pralidoxime. The in vitro pralidoxime EC(50) was estimated to be 4.67 mg/l. Animals exposed to diethylparaoxon exhibited a decrease in respiratory rate and an increase in expiratory time, and pralidoxime treatment resulted in a rapid complete but transient (< 30 min) correction in respiratory toxicity. In contrast, there was a fast and total reactivation of blood cholinesterase activity over the 210-min study period. The in vitro TD model was extended to capture the time-course of in vivo pralidoxime antidotal effects, which explained the complex relationship between drug exposure and pharmacological response profile. This study provides insights into the role of oxime-rescue of paraoxon-induced toxicity, and the final PK-TD model might prove useful in optimizing the design and development of such therapy.